Scalable Production of Adenovirus Vectors - Adenovirus: Methods and Protocols - page 175

10. Resuspend pelleted cells with 5 mL fresh Optipro™ SFM
medium.
11. Add cells suspension to the T-flask.
12. Place the T-flask in the incubator until new split (3-4 days).

Cell concentration is determined by the trypan blue exclusion
method counting cells in a hemocytometer ( see Note 3 ). Cell sus-
pensions are obtained directly from suspension cultures or after cell
detachment ( see Subheading 3.1 ) from adherent cultures.
Detachment of MDCK-E1 cells from microcarriers cultures is
performed as follows:

3.2 Monitoring
and Characterization
of Cells and AdV
Production

3.2.1 Determination of
Cell Concentration and
Viability

1. Transfer 1 mL of cell culture to an eppendorf tube and allow
microcarriers to settle-down.
2. Remove 800 μL of medium and wash cells with 800 μL PBS.
3. Remove PBS, add 800 μL Trypsin and incubate at 37 ºC during
15 min. Agitate gently the eppendorf tube during this time ( see
Note 10 )
4. Resuspend cells, let microcarriers settle-down and estimate
cell concentration and viability with trypan blue dye using a
hemocytometer.

Quantification of infectious particles of both adenoviral vectors,
done by monitoring the expression of GFP target cells ( see Note
11 ), are shown as example. HEK 293 cells and MDCK-E1 cells
used for the titration of infectious HAdV and CAV-2 vectors,
respectively, are maintained in static conditions in DMEM supple-
mented with 10 % (v/v) FBS using a similar procedure described
in Subheading 3.1.2 . The FBS present in culture medium is suffi-
cient to inactivate Trypsin, thus centrifugation step is not required
when maintaining these cells.

3.2.2 Virus Titration

1. Start with a 175 cm 2 T-flask with 80-90 % confluent HEK 293
cells ( see Note 12 ) or MDCK-E1 cells ( see Note 13 ).
2. Discard media and wash cells with 5 mL of D-PBS.
3. Discard D-PBS and add 4 mL of Trypsin-EDTA 0.05 %.
4. As soon as the cells start to detach, hit once on the side of the
T-flask to detach the cells quicker.
5. Add 6 mL of DMEM with 10 % FBS and resuspend cells.
6. Transfer 0.5 mL to an eppendorf tube for cell counting.
7. Determine cell concentration ( see Subheading 3.2.1 ).
8. Prepare a suspension of 0.25 × 10 6 HEK293 cells/mL, or
8 × 10 4 cells/mL MDCK-E1 cells in DMEM with 10 % FBS.
9. Seed cells in 24-well plates with 1 mL of this suspension per well
(0.25 × 10 6 HEK293 cells/well or 4 × 10 4 MDCK-E1 cells/well).